Use of Cobra neurotoxin polypeptide molecule in the treatment of nephropathy proteinuria

Abstract

With years of disease progression, a small amount of albumin in the blood of nephropathy patients started to leak into the urine, however very likely to be ignored due to mild clinical symptoms or confused with proteinuria caused by hypertension and/or hyperlipidemia with progress. But when other biomarkers of renal function are also found significantly increased in urine, such as β2 microglobulin α1 microglobulin, transferrin and immunoglobulin, the renal function has been impacted by pathological changes. At present, there is no effective method except hormone for nephropathy proteinuria treatment, and long-term hormone treatment will bring many side effects to patients. If the course of disease progresses freely, more protein will leak into the urine, and the renal damage will be getting worse. Cobrotoxin polypeptide molecule can effectively reduce the leakage/proteinuria, control and delay the renal pathological progression and improve renal function by inhibiting the autoimmune inflammatory response of kidney, thus contribute significantly to the treatment of nephropathy.

Claims

1. A method for treating nephropathy proteinuria in a mammal. Said method comprising administering to a mammal in need thereof a pharmaceutical composition of a therapeutically effective amount of elapidae neurotoxin, and a pharmaceutically acceptable carrier base for use in inhibiting or controlling nephropathy proteinuria.

2. According to claim (1), wherein the nephropathy proteinuria is characterized in that they are selected from the group consisting of albumin, immunoglobulins, β2 microglobulin, α1 microglobulin and transferrin, and wherein the urinary protein is over the normal level of medical diagnosis.

3. According to any claims of (1 or 2) wherein the nephropathy proteinuria is further characterized in that it comprises the increase of one or multiple, or all levels of urinary protein biomarkers, including albumin, immunoglobulins, β2 microglobulin, α1 microglobulin and transferrin.

4. According to claim (1), wherein the nephropathy proteinuria is further characterized in that they are selected from the group consisting of diabetic nephropathy proteinuria, chronic nephropathy proteinuria, acute nephropathy proteinuria, hypertensive nephropathy proteinuria, IgA nephropathy proteinuria.

5. The elapidae neurotoxin of claim (1), wherein it is an elapidae neurotoxin polypeptide having the amino acid sequence shown in SEQ ID No.1 to SEQ ID No.14; or elapidae neurotoxin polypeptide homologues having 70% or more homology with the elapidae neurotoxin polypeptide of SEQ ID No.1 to SEQ ID No.14, and the biological function of the elapidae neurotoxin polypeptide homologues is the same as or similar to that of the elapidae neurotoxin polypeptide of the amino acid sequence ID No. 1 to SEQ ID No. 14.

6. Elapidae neurotoxin polypeptides or elapidae neurotoxin polypeptides homologues according to claims (1), are further characterized in that they are derived from natural snake venoms, or synthesized from chemical polypeptides, or obtained from prokaryotic or eukaryotic hosts using recombinant technology (such as Bacteria, yeast, higher plants, insects and mammalian cells).

7. The recombinantly produced elapidae neurotoxin polypeptide or its homologues according to claim (6), based on the host used in the recombinant production scheme, the polypeptide or its homologues of the present invention may be glycosylated, or may be non-glycosylated; Disulfide-bonded or non-disulfide-bonded. The polypeptides and its homologues described in the present invention may also include or exclude the starting methionine residue.

8. The elapidae neurotoxin polypeptide as in any of claims (1, 5, 6, 7), further characterized in that the polypeptide in the present invention may include fragments of the above-mentioned various elapidae neurotoxin polypeptides after hydrolysis or enzymolysis, derivatives or analogs treated by physical, chemical or biological method, they are polypeptides which basically maintain the same biological function or activity as the above-mentioned elapidae neurotoxin polypeptide. The fragments, derivatives or analogs described in the present invention may be a polypeptide in which one or more amino acid residues are substituted, or a polypeptide having a substituent group in one or more amino acid residues, or combined with a compound (such as compounds that extend the half-life of a polypeptide, such as polyethylene glycol), or a polypeptide formed by fusion of a fatty chain, or a polypeptide formed by fusing an additional amino acid sequence to this polypeptide sequence. As described herein, these fragments, derivatives, and analogs are within the scope of those skilled in the art.

9. The method of claim (1) comprising intravenous, intramuscular, subcutaneous, intra-articular, oral, sublingual, nasal, rectal, topical, intradermal, intraperitoneal, intrathecal administration or transdermal administration.

10. The dose of elapidae neurotoxin of the method of claim (1) includes from 1 μg/Kg to 350 μg/kg each time, and the injection frequency ranges from once a day to multiple times a day, or multiple times a year.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 is 12 protein peaks of crude venom from Naja atra separated by TSK CM-650 (M) column with ammonium acetate as buffer and eluent

EXAMPLES

Example A: Obtaining neurotoxin polypeptide SEQ ID No1 from Naja atra

[0023] 1. Separation and Purification of Venom Toxins from Naja atra

[0024] Dissolve 1 g of Naja atra crude venom in 25 ml 0.025 mol ph6 0 ammonium acetate buffer, centrifugation at low temperature, and take the supernatant; Use 0.025 mole ph6 0 ammonium acetate solution to balance TSK cm-650 (m) column; After loading the sample, 0.1˜0.5 mol and 0.7˜1.0 mol, pH5.9 ammonium acetate buffer was used for elution in two compartment gradient, and the UV detection parameter was set at 280 nm; Elution flow rate: 48 ml/h; Various toxin components were collected according to the recorded spectrum, and 12 protein peaks were washed out from the collected solution.

[0025] 2. Identify the Naja atra Toxin with Affinity to Nicotinic Acetylcholine Receptor (nAChR)

[0026] The method was based on the affinity test between 12 protein peaks and nicotinic acetylcholine receptor (nAChR).

[0027] The Principle of Affinity Test

[0028] Because α-bungarotoxin has high affinity with the nicotinic acetylcholine receptor (nAChR), the cobra neurotoxin can compete with α-bungarotoxin for binding with nicotinic acetylcholine receptor, and only these α-bungarotoxin labeled with the radionuclide 125I and bound with the nicotinic acetylcholine receptor will be precipitated and be counted by the Gamma immunocount instrument whilst unbound α-bungarotoxin will be washed out. Therefore, 12 isolated proteins binding inhibition rate of α-bungarotoxin with nicotinic acetylcholine receptor (nAChR) could be used to measure the affinity between the isolated proteins and nicotinic acetylcholine receptor (nAChR), that's each isolated protein's bioactivity, which could be further determined by the γ counting value per second (Bq) measured by immune counter, and it becomes the index of proteins bioactivity.

[0029] The way of implementation include the method of binding inhibition rate of 125I radioactive labeling-α-bungarotoxin with nicotinic acetylcholine receptor (%) which reflects the activity of each protein.

[0030] The method for identifying cobra neurotoxin with high affinity with nicotinic acetylcholine receptor (nAChR) includes the following steps:

[0031] Take the above isolated snake venom protein (just one purified of 12 protein peaks each time) and rat skeletal muscle nAChR extract, 1 μL monoclonal antibody against acetylcholine nicotine receptor (mab35) 5.9 mg/ml, labeled with radionuclide 125I α-Bungarotoxin 1 μl (125I-n α-Btx) 0.18 μG/ml, after mixing, stand at 4° C. for more than 10 hours; The next day, Rabbit anti rat IgG (4.5 mg/ml) was added for 10 μL, stand at 4° C. for 2 hours; Centrifuge at 13000 rpm for 5 min, and wash the precipitate with Triton X-100 lotion for 3 times; γ count value per second (BQ) of protein activity index was measured by immunocounter; CαBTX, CBSA and C Snake Venoms refer to the BQ values of positive control αBTX, negative control BSA and isolated snake venom components respectively.

Calculation of “125I-αBtx-nAChR binding inhibitory rate (%)”=100×(CBSA−Ccbx)÷(CBSA−CαBtx),

[0032] Where

[0033] CBSA means concentration using beef serum albumin to inhibit 125I-aBtx-nAChR binding, 0% inhibited.

[0034] CaBtx means concentration using α-bungarotoxin to inhibit 125I-aBtx-nAChR binding, 100% inhibited.

[0035] Ccbx means concentration using isolated snake venom (cobrotoxin) to inhibit 125I-αBtx-nAChR binding.

[0036] The results showed that peak A was the active peak, the inhibition rate was about 50%, indicating that it had neurotoxin activity and higher affinity with nAChR. (FIG. 1)

[0037] 3. The Primary Structure Analysis of Peak A Protein-Amino Acid Sequencing

[0038] The amino acid sequencing method comprises the following steps:

[0039] Reversed phase high performance liquid chromatography (RP-HPLC) column (4.6×250 mm, VYDAC RP-C8, 5 μm) was used for purification and desalt of peak A; the N-terminal and C-terminal amino acid sequences were determined by ABI 491 protein sequence analyzer; The N-terminal amino acids obtained by sequencing were analyzed by BLSAT, and the theoretical amino acid sequence of cobra neurotoxin was predicted by comparing with the existing cobra neurotoxin sequence; the peptide coverage of cobra neurotoxin was analyzed. The experimental method was to enzymolysis the protein samples with trypsin, chymotrypsin and Glu-C enzyme respectively; Then, LC-MS/MS (xevog2 XS QTOF waters) was used to analyze the peptide samples after enzymatic hydrolysis; Use Unifi (1.8.2, waters) software to analyze LC-MS/MS data, and determine the peptide coverage of the test article according to the algorithm results. Finally, the sequence was confirmed by Edman degradation method.

[0040] The amino acid sequence of the primary structure of peak A protein obtained by sequencing is: (see amino acid sequence list <400> 1), and the corresponding amino acid sequence FASTA form is: (SEQ ID No.1). Cobra neurotoxin polypeptide (SEQ ID No.2-SEQ ID No.14) can be obtained by the same method.

Example B: Use Cobra Neurotoxin Polypeptides SEQ ID No.1 and SEQ ID No.2 for the Treatment of Adriamycin Induced Nephritis Proteinuria in Rats

[0041] 1. Experimental Animals and Groups

[0042] 80 male SD rats weighing 160-180 g were randomly divided into 2 groups: 20 in the control group and 60 in the model group. 40 rats in model group, after success in modeling, were randomly divided into cobra neurotoxin polypeptide treatment group and model group.

[0043] Each group of the three, namely control group, treatment group and model group were randomly divided into two groups again, with 10 rats in each group to test cobra neurotoxin peptide SEQ ID No. 1 and SEQ ID No. 2, the remaining rats were out of the groups.

[0044] 2. Modeling Method

[0045] Glomerulosclerotic rats were established by unilateral nephrectomy combined with tail vein injection of adriamycin twice every 2 weeks. Model. [30] rats rested for 3 days after unilateral nephrectomy, and then injected adriamycin 3 mg/kg and adriamycin 2 mg/kg from caudal vein on the 4th and 18th days respectively, the urinary protein of the surviving rats was detected 7 days after the second injection of adriamycin, the quantitative urinary protein over 100 mg/24 h suggesting the success modeling, then they were randomly divided into model group or treatment group and under observation continuously for 8 weeks. The control group was injected into caudal vein on the 4th and 18th day respectively with normal saline 0.5 ml.

[0046] During the 8 weeks of observation, the treatment group was given cobra neurotoxin polypeptide.Math.20 ug/kg by gavage once a day, continuously for 8 weeks; the model group and the control group were perfused with normal saline once a day for 8 weeks.

[0047] 3. Collection and Detection of Urinary Protein

[0048] After the last treatment, the rats were placed in the metabolic cage to collect 10 ml of urine, centrifuged at 3500 r/min for 10 min, the supernatant was extracted into the cryopreservation tube, stored in the refrigerator at −80° C. for standby, and the urinary A1 microglobulin was detected by enzyme-linked immunosorbent assay, β2-microglobulin, microalbumin, transferrin and immunoglobulin G (IgG), operate according to the instructions of ELISA kit.

[0049] Anesthetized rats were injected intraperitoneally with 1.5% Pentobarbital Sodium (50 mg/kg). After the corneal reflex disappeared, the abdominal cavity was opened and the abdominal aorta was exposed. The abdominal aorta collected 10 ml of whole blood and placed in a centrifuge tube. After standing for 2 h, centrifuged at 3500 r/min for 10 min. The supernatant was extracted in a cryopreservation tube and stored in a refrigerator at −80° C. for standby. The serum creatinine, urea nitrogen, cholesterol and triglyceride were detected by a full-automatic biochemical analyzer.

[0050] 4. Comparison of Urinary Protein Content Between Control Group, Modeling Group and Treatment Group

TABLE-US-00002 TABLE 1 TABLE-1 SEQ ID No. 1 cobrotoxin polypeptide was used for treatment group adriamycin UALB a1-MG β2-MG TRF IgG 3 + 2 mg/kg (μg/ml) (μg/ml) (ng/ml) (μg/ml) (μg/ml) control group 0.41 ± 0.08 0.29 ± 0.06 8.26 ± 1.76 0.04 ± 0.02 0.09 ± 0.04 modeling group 1.26 ± 0.23 0.61 ± 0.17 40.12 ± 11.28 0.24 ± 0.07 0.47 ± 0.16 cobrotoxin group 0.89 ± 0.11 0.52 ± 0.8  29.29 ± 6.93  0.17 ± 0.08 0.34 ± 0.08 a) The effect of cobrotoxin polypeptide on the microalbuminuria (UALB) of adriamycin induced diabetic nephropathy in rats, compared with the control group, the microalbuminuria (UALB) of the modeling group was significantly increased; compared with the modeling group, the microalbuminuria of cobrotoxin polypeptide group was significantly lower. * * It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.01). b) The effect of cobrotoxin polypeptide on α1 microglobulin (α1 mg) of Adriamycin induced diabetic nephropathy in rats, compared with the control group, α 1 microglobulin(A1 mg) of the modeling group was increased significantly; compared with the modeling group, the urine α 1 microglobulin of cobrotoxin polypeptide group decreased significantly. * * It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.01). c) The effect of cobrotoxin polypeptide on β 2 microglobulin (β 2-mg) of Adriamycin induced diabetic nephropathy in rats, compared with the control group, β 2 microglobulin (β 2-mg) of the modeling group was increased significantly; compared with the modeling group, the urine β 2 microglobulin of cobrotoxin polypeptide group decreased significantly. * * It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.05). d) The effect of cobrotoxin polypeptide on transferrin (TRF) of Adriamycin induced diabetic nephropathy in rats, compared with the control group, transferrin (TRF) of the modeling group was increased significantly; compared with the modeling group, the urine transferrin (TRF) of cobrotoxin polypeptide group decreased significantly. * * It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.05). e) The effect of cobrotoxin polypeptide on immunoglobulin (IgG) of Adriamycin induced diabetic nephropathy in rats, compared with the control group, immunoglobulin (IgG) of the modeling group was increased significantly; compared with the modeling group, the urine immunoglobulin (IgG) of cobrotoxin polypeptide group decreased significantly. * * It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.05).

TABLE-US-00003 TABLE 2 Table-2 SEQ ID No. 2 cobrotoxin polypeptide was used for the treatment group adriamycin UALB a1-MG β2-MG TRF IgG 3 + 2 mg/kg (μg/ml) (μg/ml) (ng/ml) (μg/ml) (μg/ml) control group 0.52 ± 0.13 0.34 ± 0.08 6.16 ± 1.05 0.03 ± 0.006 0.11 ± 0.04 modeling group .sup. 1.57 ± 0.19 #   1.10 ± 0.31 **   36.08 ±= 3.63 * .sup. 0.18 ± 0.087 * .sup. 0.53 ± 0.22 * cobrotoxin group 0.82 ± 0.21 0.74 ± 0.14 30.01 ±= 6.47 0.11 ± 0.032 0.31 ± 0.15 a) The effect of cobrotoxin polypeptide on the microalbuminuria (UALB) of adriamycin induced diabetic nephropathy in rats, compared with the control group, the microalbuminuria (UAlb) of the modeling group was significantly increased; compared with the model group, the microalbuminuria of cobrotoxin polypeptide group was significantly lower. ** It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.05). b) The effect of cobrotoxin polypeptide on α microglobulin (A1 mg) of Adriamycin induced diabetic nephropathy in rats, compared with the control group, α 1 microglobulin (A1 mg) of the modeling group was increased significantly; compared with the modeling group, the urine α 1 microglobulin of cobrotoxin polypeptide group decreased significantly. ** It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.01). c) The effect of cobrotoxin polypeptide on β 2 microglobulin (β 2-mg) of Adriamycin induced diabetic nephropathy in rats, compared with the control group, β 2 microglobulin (β 2-mg) of the modeling group was increased significantly; compared with the modeling group, the urine β 2 microglobulin of cobrotoxin polypeptide group decreased significantly. ** It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.05). d) The effect of cobrotoxin polypeptide on transferrin (TRF) of Adriamycin induced diabetic nephropathy in rats, compared with the control group, transferrin (TRF) of the modeling group was increased significantly; compared with the modeling group, the urine transferrin (TRF) of cobrotoxin polypeptide group decreased significantly. ** It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.05). e) The effect of cobrotoxin polypeptide on immunoglobulin (IgG) of Adriamycin induced diabetic nephropathy in rats, compared with the control group, immunoglobulin (IgG) of the modeling group was increased significantly; compared with the modeling group, the urine immunoglobulin (IgG) of cobrotoxin polypeptide group decreased significantly. ** It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.05).

Example C: Use Cobra Neurotoxin Polypeptides SEQ ID No.1 and SEQ ID No.2 for the Treatment Oft Streptozotocin (STZ)-Induced Nephritis Proteinuria in Rat

[0051] 1. Experimental Animals and Groups

[0052] The experimental rats were divided into two groups, 30 rats in each group, 10 rats in the treatment group; Modeling group 10; 10 rats in the control group, detail as follows: 80 male SD rats weighing 160-180 g were randomly divided into 2 groups: 20 in the control group and 60 in the model group. Forty rats in model group, after success in modeling, were randomly divided into cobrotoxin polypeptide treatment group and model group.

[0053] Each group of the three, namely control group, treatment group and model group were randomly divided into two groups again, with 10 rats in each group to test cobrotoxin peptide SEQ ID No. 1 and SEQ ID No. 2, the remaining rats were out of the groups. The treatment group was given cobrotoxin polypeptide.Math.20 ug/kg by gavage once a day, continuously for 8 weeks; the model group and the control group were perfused with normal saline once a day for 8 weeks.

[0054] 2. Modeling Method

[0055] The model rats were fed and fed normally. Each rat was injected intraperitoneally with 0.5 ml Freund's complete adjuvant (CFA) and intraperitoneally with streptozotocin (STZ) solution the next day. Before hand, 0.1 mmol/1 pH4 5 of citric acid buffer was prepared to 1% of the concentration and was injected intraperitoneally at 55 mg/kg. After one week, blood glucose was detected by tail vein blood. The blood glucose was maintained above 16.7 mmol/L, and the urine glucose 3+ to 4+ was regarded as a successful diabetes modeling.

[0056] 3. Collection and Detection of Urinary Protein

[0057] After the last treatment, the rats were placed in a metabolic cage to collect 10 ml of urine, centrifuged at 3500 r/min for 10 min, and the supernatant was extracted into the cryopreservation tube and kept in the refrigerator at −80° C. for standby. Urinary a 1-microglobulin β2-microsphere protein, microalbumin, transferrin and immunoglobulin G (IgG), were tested by ELISA immunosorbent assay, operate according to the instructions of ELISA kit. Anesthetized rats were injected intraperitoneally with 1.5% Pentobarbital Sodium (50 mg/kg). After disappearance of corneal reflex, the intraperitoneal cavity was opened, expose the abdominal aorta, collect 10 ml of whole blood from the abdominal aorta, place it in a centrifuge tube, stand for 2 h, centrifuge at 3500 r/min for 10 min, and then lift it. Take the supernatant into the cryopreservation tube, store it in the refrigerator at −80° C. for standby, and use the automatic biochemical analyzer to detect serum creatinine and urine nitrogen, cholesterol and triglyceride.

[0058] 4. Comparison of Urinary Protein Content Between Control Group, Modeling Group and Treatment Group

TABLE-US-00004 TABLE 3 TABLE-3 SEQ ID No. 1 cobrotoxin polypeptide was used for treatment group streptozotocin UALB a1-MG β2-MG TRF IgG (STZ) 55 mg/kg (μg/ml) (μg/ml) (ng/ml) (μg/ml) (μg/ml) control group 0.51 ± 0.10 0.61 ± 0.10 10.16 ± 2.31    0.05 ± 0.02 0.13 ± 0.06 modeling   2.21 ± 0.20 **   2.11 ± 0.32 ** 60.18 ± 15.06 ** .sup. 0.31 ± 0.19 * .sup. 0.59 ± 0.28 * cobrotoxin group 1.21 ± 0.14 1.02 ± 0.21 38.90 ± 11.82   0.17 ± 0.05 0.35 ± 0.11 a) The effect of cobrotoxin polypeptide on the microalbuminuria (UALB) of streptozotocin (STZ) - induced diabetic nephropathy in rats, compared with the control group, the microalbuminuria (UAlb) of the modeling group was significantly increased; compared with the modeling group, the microalbuminuria of cobrotoxin polypeptide group was significantly lower. ** It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.01). b) The effect of cobrotoxin polypeptide on α microglobulin (A1 mg) of streptozotocin (STZ) induced diabetic nephropathy in rats, compared with the control group, α 1 microglobulin of the modeling group was increased significantly; compared with the modeling group, the urine α 1 microglobulin of cobrotoxin polypeptide group decreased significantly. ** It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.01). c) The effect of cobrotoxin polypeptide on β 2 microglobulin (β 2-mg) of streptozotocin (STZ) induced diabetic nephropathy in rats, compared with the control group, β 2 microglobulin (β 2-mg) of the modeling group was increased significantly; compared with the modeling group, the urine β 2 microglobulin of cobrotoxin polypeptide group decreased significantly. ** It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.01). d) The effect of cobrotoxin polypeptide on transferrin (TRF) of streptozotocin (STZ) induced diabetic nephropathy in rats, compared with the control group, transferrin (TRF) increased of the modeling group was significantly; compared with the modeling group, the urine transferrin (TRF) of cobrotoxin polypeptide group decreased significantly. ** It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.05). e) The effect of cobrotoxin polypeptide on immunoglobulin (IgG) of streptozotocin (STZ) induced diabetic nephropathy in rats, compared with the control group, immunoglobulin (IgG) of the modeling group was increased significantly; compared with the modeling group, the urine immunoglobulin (IgG) of cobrotoxin polypeptide group decreased significantly. ** It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.05).

TABLE-US-00005 TABLE 4 TABLE-4 SEQ ID No. 2 cobrotoxin polypeptide was used for treatment group streptozotocin UALB a1-MG β2-MG TRF IgG (STZ) 55 mg/kg (μg/ml) (μg/ml) (μg/ml) (μg/ml) (μg/ml) control group 0.38 ± 0.12 0.53 ± 0.12 8.52 ± 1.80   0.07 ± 0.01 0.11 ± 0.04 modeling group   1.60 ± 0.29 **   2.27 ± 0.23 ** 45.21 ± 11.25 ** .sup. 0.28 ± 0.10 * .sup. 0.44 ± 0.17 * cobrotoxin group 0.89 ± 0.26 0.96 ± 0.19 30.34 ± 9.31    0.18 ± 0.06 0.31 ± 0.09 a) The effect of cobrotoxin polypeptide on the microalbuminuria (UALB) of streptozotocin (STZ) - induced diabetic nephropathy in rats, compared with the control group, the microalbuminuria (UAlb) of the modeling group was significantly increased; compared with the modeling group, the microalbuminuria of cobrotoxin polypeptide group was significantly lower. ** It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.01). b) The effect of cobrotoxin polypeptide on α microglobulin (A1 mg) of streptozotocin (STZ) induced diabetic nephropathy in rats, compared with the control group, α 1 microglobulin of the modeling group was increased significantly; compared with the modeling group, the urine α 1 microglobulin of cobrotoxin polypeptide group decreased significantly. ** It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.01). c) The effect of cobrotoxin polypeptide on β 2 microglobulin (β 2-mg) of streptozotocin (STZ) induced diabetic nephropathy in rats, compared with the control group, β 2 microglobulin (β 2-mg) of the modeling group was increased significantly; compared with the modeling group, the urine β 2 microglobulin of cobrotoxin polypeptide group decreased significantly. ** It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.01). d) The effect of cobrotoxin polypeptide on transferrin (TRF) of streptozotocin (STZ) induced diabetic nephropathy in rats, compared with the control group, transferrin (TRF) of the modeling group was increased significantly; compared with the modeling group, the urine transferrin (TRF) of cobrotoxin polypeptide group decreased significantly. ** It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.05). e) The effect of cobrotoxin polypeptide on immunoglobulin (IgG) of streptozotocin (STZ) induced diabetic nephropathy in rats, compared with the control group, immunoglobulin (IgG) of the modeling group was increased significantly; compared with the modeling group, the urine immunoglobulin (IgG) of cobrotoxin polypeptide group decreased significantly. ** It indicates that the cobrotoxin polypeptide group is compared with the modeling group (P < 0.05).

[0059] Cobrotoxin polypeptide could also reduce the indexes of blood creatinine, urea nitrogen, cholesterol and triglyceride in rats after modeling, but there was no significant difference between the treatment group and the modeling group (P>0.05). This may be because in the early stage of nephritis, the indexes of blood creatinine, urea nitrogen, cholesterol and triglyceride change mildly, and the analysis of small samples can not make statistical significance, so the decline of these indexes needs to be confirmed after further expansion of the samples.

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